8/13/2019 Mechanisms of Protein Oligomerization, The Critical Role of Insertions and Deletions in Maintaining Different Oligo

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8/13/2019 Mechanisms of Protein Oligomerization, The Critical Role of Insertions and Deletions in Maintaining Different Oligo

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important evolutionary mechanism of oligomerization, and mayprofoundly affect complex stability and the development of newspecificities. Indeed our computational experiments have demon-strated that removal of enabling regions from protein structuresresults in the complete or partial loss of dimer stability. Moreover,enabling and disabling regions may allow proteins to develop newspecific interactions and prevent undesired interactions betweenclose paralogs, thereby facilitating the separation of their func-tional pathways. This assumption is supported by analyses of

sequence and structure, amino acid composition, and by calcula-tions of aggregation propensities and free energies of dissociationof complexes. Lastly, we show how the enabling/disabling featuresmight be of practicalvalue in annotating protein oligomeric states.

Results

Identifying Mechanisms of Dimerization. We obtained 4,419 pairsof a dimer and a closest monomer from the same ConservedDomain Database (CDD) family for the full dataset and 532pairs for the nonredundant dataset (see Materials and Methods).This list contained 367 different CDD families (348 families inthe nonredundant dataset) encompassing a wide range of proteinbiological functions. About half of these families (168 families)represented enzymes and other major functional categories in-cluded regulatory, signaling, and transport proteins. We analyzed

pairs of dimers and monomers, their sequence, structural similar-ity, and energy of dimer dissociation. The sequence similaritybetween proteins in the dimeric andmonomericstates variedcon-siderably with the highest fraction of proteins sharing 90100%sequence identity between a dimer and a monomer (Fig. S1A),

which corresponds to the same proteins in different oligomericstates or cases where point mutation leads to the formation ordisruption of a dimer. Upon examination the majority of thesedimers were formed by domain swapping or through small scaleconformational changes. Point mutation can also have a consider-able effect on dimer formation and implicate many diseases (31).For example, we found point mutations of glutamate receptors

which apparently stabilize their dimer interface and reducedesensitization(1lb8-1mm6). Point mutation couldalsoshiftequi-librium between different oligomeric states inPyrococcusfuriosusmutants (1iz5-1iz4) and disrupt the salt bridge stabilizing thefunctional dimer in nuclear hormone receptors (2pin-1n46). Allthese findings were supported by experimental studies (3234).Other mechanisms observed in our set of dimers and monomersincluded:presence of common stabilizing ligands on interfacesincluding ions (ligand induced dimerization);regulation ofdimerization through posttranslational modifications includingphosphorylation and disulfide bond formation between two sub-units; and presence of insertions/deletions which favoreddimeric or monomericstates. We will discuss the latter mechanismin more detail.

Mechanism of Dimerization Through Insertions/Deletions of Protein

Regions.We analyzed differences in structures between mono-

mers and dimers to assess whether or not the gapped or unalignedresidues were more frequently located on interface vs. other typesof regions. We showed that the unaligned and gapped residues(inserted in the dimer compared to the monomer), occurredmore frequently on the interface than on the surface (P-values10e7)(Fig. S1B). This observation implies that they may playan important role in forming the dimer interface, constitutingenabling regions. In addition, we noticed that for very remotelyrelated monomers and dimers (of less than 20% identity) en-abling features might either be absent or obscured by the overallstructural differences as a result of evolutionary divergence.

We found 1,020 enabling regions and 351 disabling regionsfrom the full dataset and 108 enabling and 49 disabling regionsfrom the nonredundant dataset. Fig. 1 shows the length distribu-tion of these regions. Most regions are less than nine residues

long whereas some of the disabling regions contain up to twentyfive residues. Analysis of functional categories of families withenabling/disabling regions showed that enzyme families more fre-quently contained such regions (72 out of 168 families, binomialtestp-value < 0.003) than other families (46 out of 199 families).

Although even one extra residue on an interface might affectoligomer formation we concentrated on regions of four or moreresidues that clearly mediate or disrupt the homodimer interface.(Table S1lists manually analyzed representatives of enabling anddisabling regions of four or more residues and all regions arelisted inTable S2). All monomeric states from this table (exceptfor three entries) were confirmed using the (Inferred Biomolecu-lar Interaction Server) IBIS server (35) and dimer interfaces

were additionally verified by the NOXclass method (36). We alsoestimated the free energy of dissociation of dimers with the PISAalgorithm (Gdiss) which uses principles of chemical thermody-namics to detect macromolecular assemblies. We showed thatmany dimers were characterized by high binding affinity,Gdiss >4kcalmol [this value corresponds to standard energy of disso-ciation under the assumption that equilibrium concentrations ofdimers and monomers are equal (37)], implying that they werequite stable. Moreover, we performed the excision of enablingregions from the structures of protein dimers from the represen-tative set and recalculated the free energy of dissociation. Weshowed that removal of enabling regions disrupted the dimerformation in about half of families and destabilized the dimer

in other cases (Table S1). The effect of destabilization dependedconsiderably upon the protein family. When we examined struc-tural similarity in the aligned regions between proteins in twooligomeric states, it always turned out to be quite high (drmsd